Molecular Tools for the NPY Y1

Dimerization Probes, Vesicles, Catalytically Active Derivatives and Irreversible Ligands

Dissertation

zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.)

der Naturwissenschaftlichen Fakultät IV - Chemie und Pharmazie - der Universität Regensburg

vorgelegt von Stefan Weiß

aus Straubing

2011

Organic Chemistry, University of Regensburg.

The PhD thesis was submitted on: 27.01.2011

The colloquium took place on: 28.02.2011

Board of Examiners: Prof. Dr. A. Göpferich (Chairman) Prof. Dr. B. König (1st Referee) Prof. Dr. A. Buschauer (2nd Referee) Prof. Dr. H. Brunner (Examiner)

Especially I want to thank my supervisor Prof. Dr. Burkhard König for giving me the opportunity to perform these interesting investigations and for the useful discussions and his hints to improve my scientific work.

I also want to thank Prof. Dr. Armin Buschauer and Prof. Dr. Günther Bernhardt for the helpful discussions and the very good cooperation in the projects.

Many thanks go to the people in the departement of pharmacy:

Dr. Max Keller for the excellent collaboration, the interesting discussions and the support especially in the beginning of the thesis

Miroslaw Lopuch for introducing me in cell culture, microscopy experiments and for his cooperativeness

Melanie Kaske and Nathalie Pop for helping me with FACS techniques

Brigitte Wenzl and Elviria Schreiber for performing important cell assays for my thesis And all other people from the department of pharmacy for their collaboration

My gratitude goes to all coworkers in the department of organic chemistry:

Dr. Andreas Späth and Peter Raster for the good collaboration, the interesting discussions about chemistry and the culinary specialties served on silver plates Florian Schmidt, Florian Kinzl, Tobias “Fliesn” Lang and Andreas Müller for the collaboration in the lab

Karin Lehner for the casual goodies between the work

Dr. Rudi Vasold and Simone Strauß for HPLC investigations and measurements And all other people from the department of organic chemistry for their collaboration

Thomas Burgemeister for the assistance by the interpretation of 2D NMR spectra, Wolfgang Söllner and Joseph Kiermaier for recording mass spectra.

Thanks to Dr. Anja Lechner for assistance by FACS measurements in the department of immunology at the Universitätsklinikum Regensburg

I want to thank the DFG for financial support within the GRK 760.

And finally I want to thank my familiy for their support.

Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe; die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe des Literaturzitats gekennzeichnet.

Regensburg, __________________________

Stefan Weiß

„The game of life“, S. Weiss, 2006

„Eines Tages wird man offiziell zugeben müssen, dass das, was wir Wirklichkeit getauft haben, eine noch größere Illusion ist als die Welt des Traumes.“

Salvador Dali

Für meine Familie

und Jeannine

1 N

-Acyl-argininamides as NPY Y

Receptor Antagonists:

Influence of Stucturally Diverse Acyl Substituents on Stability and Affinity

1.1 Introduction

1

1.2 Results and Discussion

2

1.2.1 Synthesis of the N

-acylated argininamides 2 1.2.2 Stability and Y

R antagonistic activity 10

1.3 Conclusion

10

1.4 Experimental

11

1.4.1 General experimental conditions 11

1.4.2 Synthetic protocols and analytical data 12 1.4.3 Experimental determination of logD values

with HPLC 30

1.4.4 Investigation of stability 30

1.4.5 Radioligand binding assay 31

1.4.6 Fura-2 assay on HEL cells 31

1.4.7 Data processing 31

1.5 References

32

2 Pyrene Labeled Neuropeptide Y

Receptor Antagonists for the Detection of Y

Receptor Homodimers

2.1 Introduction

34

2.2 Results and Discussion

35

2.2.1 Synthesis and binding affinity of the pyrene

labeled Y

R antagonists 35

2.2.2 Optical properties of the pyrene-labeled

compounds 39

2.2.3 Fluorescence microscopy on MCF-7 cells 41

2.2.4 FACS analysis of MCF-7 cells 42

2.3 Conclusion and Outlook

43

2.4 Experimental

44

2.4.1 General experimental conditions 44

2.4.2 Synthetic protocols and analytical data 45

2.4.3 Determination of the quantum yield 49

2.4.4 MCF-7 cell culture and fluorescence

microscopy 50

2.4.5 FACS analysis of MCF-7 cells 51

2.5 References

51

3.1 Introduction

53

3.2 Results and Discussion

54

3.2.1 Synthesis of the lipophilic Y

R antagonists

and dyes 54

3.2.2 Y

R affinity of the lipophilic compounds 55

3.2.3 Formulation of the liposomes and studies

on MCF-7 cells 56

3.3 Conclusion and Outlook

59

3.4 Experimental

60

3.4.1 General experimental conditions 60

3.4.2 Synthetic protocols and analytical data 61

3.4.3 Vesicle preparation 62

3.4.4 Size exclusion chromatography 62

3.4.5 Cell culture and confocal microscopy 62

3.5 References

63

4 Towards the Catalytic Staining of NPY Y

Receptors on Living MCF-7 Cells with DMAP Modified BIBP 3226 Derivatives

4.1 Introduction

64

4.2 Results and Discussion

65

4.2.1 Synthesis and binding affinity of the catalytic

active Y

R antagonists 65

4.2.2 Synthesis and investigation of active esters 68

4.2.3 Acyl-transfer studies 69

4.2.4 Catalytic staining of MCF-7 cells 71

4.3 Conclusion

73

4.4 Experimental

73

4.4.1 General experimental conditions 73

4.4.2 Synthetic protocols and analytical data 74 4.4.3 Radioligand competition binding assay 81

4.4.4 Fura-2 assay on HEL cells 81

4.4.5 Active ester kinetics 81

4.4.6 Acyl-transfer studies 81

4.4.7 Cell culture and confocal microscopy 82

4.4.8 Data processing 82

4.5 References

83

5.1 Introduction

85

5.2 Results and Discussion

89

5.2.1 Synthesis 89

5.2.2 Fluorescence based ligand binding studies 89 5.2.3 Stucture and irreversible binding properties 91 5.2.4 Radioligand binding studies 91 5.2.5 Functional studies on HEL cells 92

5.3 Conclusion

94

5.4 Experimental

94

5.4.1 General experimental conditions 94

5.4.2 Synthetic protocols and analytical data 95

5.4.3 Radioligand competition binding assay 104

5.4.4 Fura-2 assay on HEL cells 104

5.4.5 Cell culture and confocal microscopy 104

5.4.6 Data processing 105

5.4.7 References 105

A Appendix

A.2 Curriculum Vitae 110

A.3 Publications, Posters, Professional Training 111

A.4 Summary in German and English 112

N

-Acyl-argininamides as NPY Y

Receptor Antagonists:

Influence of Stucturally Diverse Acyl Substituents on Stability and Affinity

1.1 Introduction

G Protein-coupled receptors (GPCRs) represent a major class of biological targets in drug discovery. Focusing on neuropeptide Y (NPY) and histamine receptors as models of aminergic and peptidergic GPCRs, respectively, we are particularly interested in bioisosteric approaches to develop special receptor subtype-selective tools, e. g., bivalent ligands, prodrugs, radiolabeled and fluorescent compounds.^1-6 Strongly basic groups such as guanidines (including Arg residues) are essential structural features of numerous GPCR ligands, but are unfavourable with respect to bioavailability and brain penetration. This is also true for many high affinity NPY and histamine receptor agonists and antagonists.^{7, 8} Therefore, special effort was put into the search for bioisosteric replacements of strongly basic functional groups. Recently, we reported on the exploitation of guanidine - acylguanidine bioisosterism with respect to histamine H₂, H₃ and H₄ receptor ligands^{5, 9-12} and arginine-type NPY Y₁ receptor antagonists.1, 3, 4, 6, 7, 13, 14

NPY is a highly conserved peptide which plays an important role as a neurotransmitter in the central and peripheral nervous system.¹⁵ In humans, four receptor subtypes, referred to as NPY Y1, Y2, Y4 and Y5 receptors, mediate the biological effects of NPY. For instance, in the periphery NPY Y1 receptor (Y1R) stimulation causes an increase in blood pressure. In the central nervous system (CNS) Y1R activation elicits anxiolytic and sedative effects and is involved in the stimulation of food intake.¹⁵

Arginine derivatives such as the Y1R antagonist BIBP3226¹⁶ (1a, Figure 1) have been proven as valuable pharmacological tools regardless of properties being far from drug-like.

Interestingly, the reduction of the basicity of the Arg-derived Y1R antagonist 1a by introducing electron-withdrawing N^G-substituents such as acyl groups turned out to be a promising general route to guanidine derivatives with increased potency, to fluorescent ligands and radioactive tracers.^{3-7, 14} Even bulky fluorophores attached to the guanidine via spacers of different size were tolerated; a moderate decrease in affinity did not compromise suitability of the compounds as pharmacological tools. However, depending on the chemical

nature of the linker cleavage of the acylguanidine group may occur as demonstrated for a model compound.¹⁷

In continuation of our studies on the structure-activity relationships of argininamide-type Y1R antagonists we synthesized a series of N^G-acylated analogues of 1a in order to explore the impact of structurally diverse substituents on stability and pharmacological activity in vitro (Y1R binding and antagonism). As the small library of Y1R antagonists was synthesized to cover a wide range of distribution coefficients (logD) the BIBP 3226 skeleton was substituted with hydrophobic alkyl chains or sugars and amines to alter the polarity of the parent compound (Figure 1).

1.2 Results and Discussion

1.2.1 Synthesis of the N^G-acylated argininamides

The N^G-acylated argininamides were efficiently prepared according to the general synthetic route shown in Scheme 1. N-Boc-S-methylisothiourea (3) was acylated with the respective carboxylic acids yielding the isothiourea derived guanidinylation reagents 4b-4g, 4i, 4k-4q, 4s-4u. Guanidinylation of D-ornithinamide 5 and subsequent removal of protecting groups gave the envisaged N^G-acylated argininamides. Amine 5 is available from D-ornithine in six steps in 32% overall yield.⁴

Synthetic strategies for the preparation of carboxylic acids 7, 8, 11, 14, 17, 20, 22, 27, 28, 30, and 36 are depicted in Schemes 2 – 4. Acids 7 and 8, containing a triazole entity, were prepared through a Cu(I)-catalysed “click-reaction” between 4-azidobutanoic acid and ^tBu- protected propargyl alcohol or Boc-protected propargyl amine (Scheme 2). Carboxylic acid 14 was prepared via guanidinylation of 6-aminohexanoic acid methyl ester with N,N’-diBoc- S-methylisothiourea followed by ester hydrolysis (Scheme 2). For the synthesis of succinic acid derivative 17 ethylenediamine was one-fold guanidinylated with N,N’-diBoc-S-methyl- isothiourea yielding amine 16, which was treated with succinic anhydride (Scheme 2). Acid precursors 20 and 22 were obtained through the treatment of amines 19 and 21 with succinic anhydride (Scheme 2). The suberic acid derivatives 27 and 28 were prepared through amidation of octandioic acid mono-benzyl ester with amines 24 and 21, respectively, followed by benzyl ester cleavage (Scheme 3). Octandioic acid derivative 30 was obtained via amidation of non-protected suberic acid with 3-aminopropanoic acid benzyl ester (Scheme 3).

The synthesis of carboxylic acid 36 started from pyrene-1-carboxylic acid (31), which was amidated with mono-Boc-protected ethylenediamine (21) in moderate yield (Scheme 4). The

Boc group was removed with hydrochloric acid yielding amine 33, which was coupled with 3,6,9-trioxaundecandioic acid mono-benzyl ester (34) to give compound 35. Hydrogenolysis of 35 resulted in acid 36 (Scheme 4).

Figure 1. Structures of N^G-acylated derivatives of the Y1R antagonist BIBP 3226 (1a) ranked according to calculated logD values (ACD-labs software version 12, pH = 7.4); experimental logD values from HPLC measurements¹⁸ in parentheses.

D-Gluconic acid derivative 1i was synthesized from penta-acetylated D-gluconic acid.

Unfortunately, deprotection of the hydroxyl groups failed. The base-labile acylguanidine moiety prevents basic selective cleavage of the acetyl protecting groups. Enzymatic deprotection using a lipase preparation (Novozym 435) was also unsuccessful. To circumvent

such problems, sugar derivative 1n, containing a galactose entity, was prepared. The synthesis of the respective carboxylic acid 11 is shown in Scheme 2. Two acetal protecting groups were introduced in the first step and the remaining primary hydroxyl function was esterified with succinic anhydride. The acetal groups are compatible with the general synthetic strategy outlined in Scheme 1 and can be easily removed in the final deprotection step with TFA/DCM.

BIBP 3226 derivatives 1j and 1r were prepared through 4-fluorobenzoylation and propionylation of amines 1s and 1u, respectively (Scheme 5).

Scheme 1. General route for the synthesis of N^G-acylated BIBP 3226 derivatives 1b-1g, 1i, 1k-1q and 1s-1u. a) Boc2O, NaOH, ^tBuOH, 90%; b) DIPEA, EDC, HOBT, DCM, (1s: TBTU, DIPEA, DMF), 31 - 95%; c) HgCl2, NEt3, DMF, 39 - 78%; d) TFA/DCM 1:1, quantitative yield.

Scheme 2. Synthesis of carboxylic acids 7, 8, 11, 14, 17, 20 and 22. a) CuSO₄ (5 mol%), Na- ascorbate (10 mol%), MeOH, H₂O, 67%-86%; b) acetone, iodine, 71%; c) succinic anhydride, DMAP, NEt₃, 73%; d) HgCl₂, NEt₃, DMF, 84%; e) THF, NaOH, 71%; f) CHCl₃, 16 h, 97%;

g) THF, NEt3, 49%; h) CF3COOEt, DCM; i) Boc2O, DCM; j) K2CO3, MeOH, H2O, 47%

overall; k) succinic anhydride, THF, NEt₃, 77%; l) DCM, 99%.

Scheme 3. Synthesis of suberic acid derivatives 27, 28 and 30. a) DCM, EDC, HOBt, DIPEA, 62%; b) Pd-C, H2, 90% - 94%; c) DCM, EDC, HOBt, DIPEA, 44%.

Scheme 4. Synthesis of the pyrene-1-carboxamide derivative 36. a) DCM, EDC, HOBt, DIPEA, 52%; b) MeOH, HCl, 83%; c) DMF, EDC, HOBt, DIPEA, 59%; d) Pd-C, MeOH, quantitative.

Scheme 5. Synthesis of the N^G-acylated BIBP 3226 derivatives 1j and 1r. a) NEt₃, DMF, 65%

(1j), 21% (1r).

1.2.2 Stability and Y1R antagonistic activity

As the acylguanidine moiety of N^G-acylated argininamide-type Y₁R antagonists may be considered as the most probable cleavage site the stability of the presented BIBP 3226 (1a) derivatives was investigated with respect to the formation of 1a at physiological pH of 7.4. A

release of 1a under the conditions of the pharmacological assays has to be taken into account as 1a is a highly potent Y1R antagonist (Ki = 1.3 nM) and could pretend a higher potency of the investigated compounds. Therefore the stability of the N^G-acylated argininamides was investigated at pH 7.4 on the time scale of the assays. Formation of 1a was not observed for the strongly hydrophobic compounds 1c, 1d and 1e, which are devoid of hetero atoms in their N^G-acyl substituents. The hydrophobic ligands 1f and 1g, both containing an amide group in the acyl substituent, showed a very minor decomposition over 90 min (< 0.5%). Instability was more pronounced (decomposition up to 2.6% over 90 min) for the more polar compounds (1h, 1i, 1k – 1q) bearing amide, ester, triazole, sugar, amine, carboxylic or guanidine functions in the acyl residue.

Exceptionally high instabilities were found for argininamides with succinyl attached to the guanidine (1j, 1r – 1u). Compounds 1r – 1u quantitatively decomposed to 1a over 90 min (Table 1). This process was exemplarily explored by RP-HPLC-MS using the hydrophobic compound 1j, which allows the detection of the acyl substituent upon cleavage. As becomes obvious from Figure 2 compound 1j is cleaved via an intramolecular attack of the succinic amide nitrogen at the carbonyl group attached to the guanidine resulting in the N-alkyl succinimide derivative 39. The lability of the acylguanidines bearing nucleophiles in position 5 or 6 of the acyl side chain is in accordance with the recently reported reactivity of 5- aminopentanoyl substituted guanidine.¹⁷

All compounds proved to be stable at acidic pH (0.1 % aqueous trifluoroacetic acid, pH 2-3).

Under these conditions the acylguanidine group is almost quantitatively protonated due to pKa

values in the range of 7 – 8. Obviously, this changes the susceptibility against hydrolysis and prevents intramolecular attacks of nucleophiles at the carbonyl group, respectively.

Figure 2. Exploration of the decomposition mechanism of BIBP 3226 derivative 1j incubated in an aqueous buffer (pH 7.4) at 20 °C. LC-MS analysis was performed after an incubation period of 20 and 90 min. 1j is decomposed to BIBP 3226 (1a) and the succinimide derivative 39 through an intramolecular attack of the succinic amide nitrogen at the guanidine linked carbonyl group. Chromatograms were acquired by single ion monitoring analysis.

Except for the highly unstable compounds 1j and 1r – 1u the N^G-acylated argininamides were characterised in terms of Y1R antagonism (Kb values) and Y1R affinity (Ki values) using a Fura-2 assay on human erythroleukemia (HEL) cells¹⁹ and a radioligand binding assay on SK- N-MC human neuroblastoma cells,⁴ respectively. Kb and Ki values are summarized in Table 1.

Except for 1h (Ki = 0.9 nM) all N^G-acylated argininamides showed a reduced Y1R affinity compared to the parent compound 1a (Ki = 1.3 nM, Table 1). The decrease in affinity was most pronounced (up to three orders of magnitude) for the most hydrophobic compounds (1b – 1e, Table 1), which have limited solubility and might interact with the cell membrane.

Direct attachment of pyrene-1-carboxylic acid to the guanidine-N (compound 1e) resulted in complete loss of Y₁R affinity. By contrast, high Y₁R affinity can be retained, if the pyrene

moiety is attached to the guanidine group through a linker (compounds 1d and 1f, Figure 1, Table 1).

The Y1R affinity of the polar sugar and guanidine substituted compounds 1n and 1q is about 35 times lower compared to the affinity of 1a. The argininamide 1a may be considered a mimic of the C-terminal dipeptide in NPY (...-Arg³⁵-Tyr³⁶-NH2). It may be speculated that the introduction of a second guanidine moiety (as provided by compound 1q) could enhance binding affinity through mimicking the second arginine residue (Arg³³).²⁰ However, the Ki

value of compound 1q was only 51 nM – perhaps due to an inappropriate distance between the two guanidine groups.

The hydroxy substituted Y₁R antagonist 1l (K_i = 3.0 nM), proved to be as potent as 1a. It is conceivable that the hydroxy group mimics Thr³² or Tyr²⁷ of the natural ligand NPY. Both amino acids are very important for the binding of NPY to the Y₁R, as identified by alanine scan.²¹ The amine analogue of 1l, compound 1m, binds with similar affinity to the Y₁R (K_i = 6.7 nM).

As partial decomposition of compounds 1i, 1k and 1o - 1q occurs (up to 2.6% over 90 min), the determined K_i values of these five compounds have to be considered with reservation, as the affinity of the cleavage product 1a (Ki = 1.3 nM) and the assay periods (from preparation of solutions to read out: 60 – 90 min) have to be taken into account.

With respect to the prediction of the affinity of new compounds, three issues have to be taken into account: substitution with bulky groups directly at the BIBP 3226 backbone results in a complete loss of affinity, that can be compensated with a spacer of adequate length.

Derivatives with a small aromatic or heteroaromatic substituent (1g, 1l, 1m) show slightly higher binding affinity compared to the more flexible derivatives (1g vs. 1k, 1o, 1p; 1m vs.

1q). Relatively small hydrophobic substituents, as in 1h (Ki = 0.9 nM) and the radioligand [³H]-UR-MK114 (Ki = 1.3 nM),⁴ result in similar and even higer affinity compared to BIBP 3226. Interactions of the acyl substituent with the Y1R are difficult to predict, because these residues are presumably oriented toward flexible extracellular loop regions⁷ and, depending on chemical nature and size of the acyl residue, an interaction with membrane lipids cannot be ruled out.

Table 1. LogD values, stability, Y1R antagonistic activity (Kb) and Y1R binding affinity (Ki) of BIBP 3226 derivatives 1b – 1u.

no. logD^a % decomposition (pH 7.4, 20 °C) after 20 /

90 min

Y1R antagonism K_b^b [nM]

Y1R affinity K_i ^c [nM]

1a^d 0.7 (1.4) -- 1.5 ± 0.2 1.3 ± 0.2

1b 12.5 n.d. 140 ± 10 1500 ± 120

1c 9.4 0 / 0 170 ± 8 460 ± 19

1d 7.6 (5.3) 0 / 0 n.d.^e 270 ± 70

1e 7.2 0 / 0 n.d.^e inactive

1f 6.0 (3.1) 0 / < 0.5 n.d.^e 59 ± 16

1g 4.6 (3.2) 0 / < 0.5 1.10 ± 0.04 40 ± 9

1h 3.9 0 / 0.8 0.06 ± 0.01^f 0.9 ± 0.1^f

1i 3.5 (2.9) 0.9 /2.6 83 ± 8 110 ± 38

1j 3.5 30 /69 n.d.^g n.d.^g

1k 2.9 0 /0.7 170 ± 18 260 ± 82

1l 1.8 (1.4) 0.9 /2.1 0.40 ± 0.03 3.0 ± 0.5

1m 1.7 0.6 / 1.7 14 ± 4 6.7 ± 0.1

1n 0.9 (1.0) < 0.5 /1.3 510 ± 190 41 ± 5

1o 0.7 < 0.5 /1.4 450 ± 52 73 ± 11

1p -0.3 (1.3) < 0.5 /1.4 230 ± 64 130 ± 10

1q -0.3 (1.4) < 0.5 /1.6 18 ± 8 51 ± 18

1r -0.6 64 /100 n.d.^g n.d.^g

1s -1.3 51 /100 n.d.^g n.d.^g

1t -1.4 61 /100 n.d.^g n.d.^g

1u -1.8 (1.3) 40 /100 n.d.^g n.d.^g

a Calculated with ACD-labs software version 12, pH = 7.4; in brackets: experimental logD determined with HPLC measurements. ^b K_b values for inhibition of NPY (10 nM) induced calcium mobilization in HEL cells (Fura-2 assay); all mean values ± SEM from two or three (1k, 1n, 1o, 1p, 1u) independent experiments. ^c Kivalues determined from the displacement of 1.5 nM [³H]-UR-MK114⁴ on SK-N-MC cells; all mean values ± SEM from two or three (1c, 1f, 1g, 1k, 1l, 1p) independent experiments. ^d BIBP 3226. ^e Due to their fluorescent properties pyrene ligands are not compatible with the Fura-2 assay. ^f Keller et al.^{1 g} not determined due to the high instability.

1.3 Conclusion

The stability of the N^G-acylated argininamides strongly depends on the nature of the acyl residue. It becomes obvious from the high instability of the succinyl derivatives (1j, 1r – 1u) that cleavage is favoured when the residues harbour nucleophilic functional groups capable of an intramolecular attack on the acyl carbonyl. This is also conceivable for argininamides decomposing to a minor extent and bearing N^G-acyl residues containing amide, ester, triazole, sugar, amine, carboxylic or guanidine functions (1h, 1i, 1k – 1q). This has to be taken into account in pharmacological investigations, in particular, when cleavage products are highly bioactive as in the case of 1a (Ki = 1.3 nM). However, despite these limitations broad

structural variation of N^G-acyl substituents was tolerated (affinities in the nM range). Most of the investigated compounds proved to be sufficiently stable at pH 7.4 to determine reliable in vitro pharmacological data. In conclusion, the N^G-acylation of the stongly basic guanidine group in argininamides is a successful bioisosteric approach to more drug-like properties provided that the outlined stability considerations are taken care of. This is not restricted to the NPY field, as guanidine groups are crucial structural features of many different biologically active compounds including GPCR ligands

1.4 Experimental

1.4.1 General experimental conditions

Unless otherwise noted, solvents (analytical grade) were purchased from commercial suppliers and used without further purification. Ethyl acetate (EA), petrol ether (PE, 60 – 70

°C), methanol and dichloromethane were obtained in technical grade and distilled before application. Acetonitrile (MeCN) for HPLC was obtained from Merck (Darmstadt, Germany).

Pentacosa-10,12-diynoic acid (Sigma-Aldrich Chemie GmbH, Munich, Germany), 4-(pyren- 1-yl)butanoic acid (Sigma-Aldrich Chemie GmbH, Munich, Germany), hexadecanoic acid (Riedel-de Haen, Seelze, Germany), suberic acid (Fluka, Sigma-Aldrich Chemie GmbH, Munich, Germany) and D-(+)-galactose (Merck, Darmstadt, Germany) were purchased.

Preparative HPLC was performed with a system from Knauer (Berlin, Germany) consisting of two K-1800 pumps and a K-2001 detector. A Nucleodur 100-5 C18 (250 × 21 mm, 5 µm;

Macherey-Nagel, Germany) and a Eurospher-100 C18 (250 × 32 mm, 5 µm; Knauer, Germany) served as RP-columns at flow rates of 20 and 38 mL/min, respectively. Mixtures of MeCN and diluted aqueous TFA (0.1 %) were used as mobile phase. ¹H-NMR spectra were recorded at 300 MHz on a Bruker Avance 300 spectrometer or at 600 MHz on a Bruker Avance III 600 with cryogenic probehead (Bruker, Karlsruhe, Germany). ¹³C-NMR spectra were recorded at 75 MHz on a Bruker Avance 300 spectrometer. All chemical shifts values are reported in ppm. UV/VIS spectra were recorded with a Varian Cary BIO 50 UV/VIS/NIR spectrophotometer (Varian Inc., CA, USA). Mass spectra: Finnigan SSQ 710A (EI), Finnigan MAT 95 (CI), Finnigan MAT TSQ 7000 (Thermo FINNIGAN, USA) (ES/LC-MS). LC- system for LC-MS: Agilent 1100 (Palo Alto, USA). LC-MS method I (LC-MS-I): Column:

Phenomex Luna C18, 3.0 µm, 100 x 2 mm HST (Phenomenex, Aschaffenburg, Germany);

flow: 0.30 mL/min; solvent A (water + 0.1% TFA), solvent B (MeCN); gradient: 0 min [A/B 95/5], 1 min [A/B 95/5], 11 min [A/B 2/98], 18 min [A/B 2/98], 19 min [A/B 95/5], 24 min

[A/B 95/5]. LC-MS method II: Column: Phenomex Luna C18, 2.5 µm, 50 x 2 mm HST (Phenomenex, Aschaffenburg, Germany); flow: 0.40 mL/min; solvent A (water + 0.1% TFA), solvent B (MeCN); gradient: 0 min [A/B 95/5], 8 min [A/B 2/98], 11 min [A/B 2/98], 12 min [A/B 95/5], 15 min [A/B 95/5]. Analytical HPLC (HPLC): Compounds 1c-1i, 1k-1q, 1t, 1u:

Phenomex Luna C18, 3.0 µm, 150 x 2 mm (Phenomenex, Aschaffenburg, Germany); flow:

0.30 mL/min; solvent A (H2O + 0.0059%TFA), solvent B (MeCN); gradient: 0 min [A/B 95/5], 30 min [A/B 2/98]; 1j, 1r, 1s: Eurospher-100 C18, 5 µm, 250 x 4.0 mm (Knauer, Berlin, Germany); flow: 0.80 mL/min; solvent A (water + 0.05% TFA), solvent B (MeCN);

gradient: 0 min [A/B 85/15], 28 min [A/B 45/55], 33 min [A/B 5/95], 40 min [A/B 5/95].

Melting points were determined with a Lambda Photometrics Optimelt MPA100 apparatus (Lambda photometrics, Harpenden, UK), they are not corrected. Thin layer chromatography (TLC) was performed on alumina plates coated with silica gel (Merck silica gel 60 F₂₄₅, thickness 0.2 mm). Column chromatography (CC) was performed with Merck Geduran SI 60 silica gel as the stationary phase.

The synthesis of 38, 1a and 1h was described elsewhere.^{1, 4} Compounds 3,² 5,⁴ 6,²² penta- acetyl gluconic acid,^{23 t}Bu-protected propargyl alcohol,^{24 t}Bu-protected propargyl amine,²⁵ 10,²⁶ 11,²⁷ 14,²⁸ 16,²⁹ 19,³⁰ 21,³¹ 23,³² 31,³³ 37⁴ were prepared according to literature pro- cedures.

1.4.2 Synthetic protocols and analytical data of 7, 8, 17, 20, 22, 25-28, 30 and 32-36 4-[4-(tert-Butoxymethyl)-1H-1,2,3-triazol-1-yl]butanoic acid (7)

4-Azidobutanoic acid (419 mg, 3.25 mmol) was mixed with Boc-protected propargyl alcohol (364 mg, 3.25 mmol) in 5 mL MeOH. Then ascorbic acid (65 mg, 0.33 mmol) dissolved in 1 mL H2O and copper sulphate pentahydrate (8 mg, 0.03 mmol) dissolved in 1 mL H2O were added and the reaction mixture was heated to reflux overnight. Next day MeOH was evaporated completely and dichloromethane (30 mL) and sat. aqueous NaHSO4 solution (20 mL) were added. The organic layer was collected and then diluted NaOH (40 mL, 1 mol/L) was added. The aqueous layer was collected and acidified with NaHSO4 solution until pH < 2.

Dichloromethane (40 mL) was added and the acid was extracted. The organic phase was dried over MgSO4, the solvent was evaporated and a white solid was obtained (674 mg, 86%), m.p.

81 °C. ¹H NMR (300 MHz, CDCl3): 1.26 (s, 9H), 2.15 – 2.25 (m, 2H), 2.39 (t, J = 6.88, 2H), 4.42 (t, J = 6.94, 2H), 4.58 (s, 2H), 7.55 (s, 1H), 10.31 (bs, 1H). ¹³C NMR (75 MHz, CDCl3):

25.3, 27.5, 30.5, 49.3, 56.2, 74.1, 122.5, 146.8, 176.4. C₁₁H₁₉N₃O₃: MS (CI, NH3): m/z(%) 242(100, MH+).

4-{4-[(tert-Butoxycarbonylamino)methyl]-1H-1,2,3-triazol-1-yl}butanoic acid (8)

4-Azidobutanoic acid (0.89 g, 6.90 mmol) was mixed with Boc-protected propargylamine (1.07 g, 6.90 mmol) in MeOH (10 mL). NaOH (1 mol/L) was added until the pH value was between 6 and 8. Then ascorbic acid (65 mg, 0.33 mmol) dissolved in 1 mL H2O and copper sulphate pentahydrate (8 mg, 0.03 mmol) dissolved in 1 mL H2O were added and the reaction mixture was heated to reflux overnight. Next day MeOH was evaporated under reduced pressure and the residue was diluted with 80 mL EA and 80 mL of aqueous NaHSO4 solution (5%w). The organic layer was separated, dried over MgSO4 and the solvent was evaporated.

The crude material was recrystallised from EA yielding a white crystalline solid (1.31 g, 67%), m.p. 117 °C. ¹H NMR (300 MHz, CD3OD): 1.43 (s, 9H), 2.10 – 2.20 (m, 2H), 2.25 – 2.35 (m, 2H), 4.29 (s, 2H), 4.44 (t, J = 6.89, 2H), 7.82 (s, 1H). ¹³C NMR (75 MHz, CD₃OD):

26.7, 29.0, 31.5, 36.9, 50.6, 80.5, 124.2, 147.3, 158.3, 176.1. C12H20N4O4: MS (LC-MS-II):

m/z(%) [t_R = 5.1 min]: 285(35, MH+), 569(100).

6-(tert-Butoxycarbonylamino)-2,2-dimethyl-4,11-dioxo-3-oxa-5,7,10-triaza-tetradec-5- en-14-oic acid (17)

Compound 16 (300 mg, 0.99 mmol) and succinic anhydride (119 mg, 1.19 mmol) were dissolved in a mixture of THF (10 mL) and NEt3 (150 mg, 1.49 mmol) and stirred overnight.

Next day water was added (10 mL) and the mixture was stirred for one hour. THF was evaporated completely and the residue was diluted with water (20 mL). Aqueous NaHSO4

solution (5%w, 10 mL) was added and the mixture was extracted with dichloromethane (2 x 50 mL). The organic layer was dried over MgSO4 and the solvent was evaporated yielding a white solid (197 mg, 49%), m.p. 77-80 °C. ¹H NMR (300 MHz, CDCl3): 1.47 (s, 9H), 1.49 (s, 9H), 2.50 – 2.60 (m, 2H), 2.60 – 2.70 (m, 2H), 3.35 – 3.45 (m, 2H), 3.45 – 3.60 (m, 2H), 7.00 – 8.40 (bs, 1H), 8.50 (bs, 1H), 8.67 (bs, 1H), 11.38 (bs, 1H). ¹³C NMR (75 MHz, CDCl3):

28.0, 28.2, 30.3, 30.5, 40.3, 41.9, 79.9, 83.9, 153.0, 157.6, 162.5, 172.9, 175.1. C17H₃₀N₄O₇: MS (LC-MS-I): m/z(%) [tR = 10.3 min]: 403(100, MH+), 805(10).

4-{2-[(2-tert-Butoxycarbonylaminoethyl)(tert-butoxycarbonyl)amino]ethylamino}

-4-oxobutanoic acid (20)

Compound 19 (1.00 g, 3.30 mmol) was dissolved in 5 mL THF and a solution of succinic anhydride (0.33 g, 3.30 mmol) in 5 mL THF was added. Then NEt₃ (0.50 g, 4.95 mmol) was added to the mixture. The mixture was stirred overnight at ambient temperature. Next day THF was removed completely and the crude product was dissolved in 20 mL

dichloromethane. It was washed with aqueous NaHSO4 sol. (5%w, 30 mL), dried over MgSO4 and the solvent was evaporated giving a white solid (1.03 g, 77%), m.p. 90 °C. ¹H NMR (300 MHz, CDCl3): 1.40 (s, 9H), 1.43 (s, 9H), 2.48 (bs, 2H), 2.64 (bs, 2H), 3.10 - 3.40 (m, 8H), 4.95 – 5.25 (m, 1H), 7.00 – 7.30 (m, 1H), 9.31 (bs, 1H). ¹³C NMR (75 MHz, CDCl3): 20.4, 29.7, 30.6, 39.3, 39.6, 47.0, 47.8, 79.6, 80.7, 156.6, 156.7, 172.8, 175.7.

C₁₈H₃₃N₃O₇: MS (ES): m/z(%) 204(5), 245(45), 304(50), 348(25), 404(100, MH+), 426(5), 808(7), 825(15).

3-(2-tert-Butoxycarbonylaminoethyl)aminocarbonylpropanoic acid (22)

Succinic anhydride (0.41 g, 4.06 mmol) and NEt3 (56 µL, 0.41 mmol) were added to a solution of amine 21 (0.65 g, 4.06 mmol) in dichloromethane (3 mL). The mixture was heated to 60 °C for 25 min (microwave synthesizer (Biotage Initiator 8)). After removal of the solvent under reduced pressure the product (insoluble in dichloromethane) was afforded as a white solid (1.05 g, 99%). ¹H NMR (300 MHz, DMSO-d6): 1.37 (s, 9H), 2.25 – 2.32 (m, 2H), 2.37 – 2.44 (m, 2H), 2.91 – 2.99 (m, 2H), 3.00 – 3.08 (m, 2H), 6.78 (t, J = 5.45, 1H), 7.85 (t, J = 5.29, 1H), 12.08 (bs, 1H). ¹³C NMR (75 MHz, DMSO-d6): 28.1, 29.0, 29.9, 38.6, 39.5, 77.5, 155.5, 171.0, 173.8. C11H20N2O5: (LC-MS-II): m/z(%) [tR = 4.63 min]: 261(40, MH+), 278(6, MNH4+), 521(35, 2MH+), 538(100, 2MNH4+).

Benzyl 8-(3-methoxy-3-oxopropylamino)-8-oxooctanoate (25)

Acid 23 (250 mg, 0.95 mmol), DIPEA (368 mg, 2.85 mmol), and HOBT ⋅ H2O (142 mg, 1.05 mmol) were dissolved in ice-cold dichloromethane (20 mL) and EDC (147 mg, 0.95 mmol) was added under nitrogen atmosphere. After 15 minutes amine 24 (98 mg, 0.95 mmol) was added. The reaction mixture was stirred overnight. Next day the reaction mixture was washed with aqueous NaHSO4 sol. (5%w, 20 mL) and sat. aqueous NaHCO3 solution (20 mL). The solvent was dried over MgSO4, filtered and evaporated. The crude product was purified by column chromatography (PE/EA 3:1 -> EA Rf = 0.4 [EA]) yielding the product as a white solid (253 mg, 76%), m.p. 48 °C. ¹H NMR (300 MHz, CDCl3): 1.31 (m, 4H), 1.60 (m, 4H), 2.10 (m, 2H), 2.30 (t, J = 7.48, 2H), 2.50 (m, 2H), 3.50 (dd, J1 = 6.07, J2 = 11.99, 2H), 3.68 (s, 3H), 5.10 (s, 2H), 6.00 – 6.10 (bs, 1H), 7.30 – 7.38 (m, 5H). ¹³C NMR (75 MHz, CDCl3):

24.0, 25.4, 28.75, 28.80, 33.9, 34.2, 34.7, 36.6, 51.8, 66.1, 128.2, 128.6, 136.1, 173.0, 173.2, 173.6. C19H27NO5: MS (CI, NH3): m/z(%) 350(100, MH+).

Benzyl 8-[2-(tert-butoxycarbonylamino)ethylamino]-8-oxooctanoate (26)

Compound 23 (264 mg, 1.00 mmol) was dissolved in 10 mL of dichloromethane. HOBT ⋅ H2O (149 mg, 1.10 mmol) and DIPEA (387 mg, 3.00 mmol) were added under nitrogen atmosphere. The mixture was stirred and cooled in an ice bath. EDC (171 mg, 1.10 mmol) was added and after 15 min amine 21 (160 mg, 1.00 mmol) was added. The mixture was stirred overnight. Next day DCM was added (10 mL), the organic phase was washed once with aqueous NaHSO4 (5%w, 20 mL) and sat. aqueous NaHCO3 sol. (20 mL), was dried over MgSO4 and the organic layer was evaporated. The crude product was purified by column chromatography (PE/EA 1:1 -> EA Rf = 0.3 [EA]). A white, wax-like solid (251 mg, 62%), m.p. 60 °C was obtained. ¹H NMR (300 MHz, CDCl3): 1.25 – 1.25 (m, 4H), 1.43 (s, 9H), 1.55 – 1.70 (m, 4H), 2.10 – 2.17 (m, 2H), 2.30 – 2.37 (m, 2H), 3.20 – 3.30 (m, 2H), 3.30 – 3.40 (m, 2H), 4.97 (bs, 1H), 5.10 (s, 2H), 6.20 (bs, 1H), 7.30 – 7.40 (m, 5H). ¹³C NMR (75 MHz, CDCl₃): 24.7, 25.4, 28.4, 28.7, 28.8, 34.1, 36.5, 40.3, 40.7, 66.1, 79.6, 128.2, 128.6, 136.1, 157.0, 173.6, 173.8. C22H34N2O5: MS (ES): m/z(%) 407(100, MH+).

8-(3-Methoxy-3-oxopropylamino)-8-oxooctanoic acid (27)

Compound 25 (210 mg, 0.60 mmol) was dissolved in MeOH, placed into an autoclave and Pd/C (25 mg) was added after flushing the autoclave with N₂. The reaction mixture was stirred overnight at room temperature and 12 bar hydrogen pressure. Pd/C was removed by filtration over celite, MeOH was evaporated and a white solid was obtained (146 mg, 94%), m.p. 73 °C. ¹H NMR (300 MHz, CD3OD): 1.34 (m, 4H), 1.59 (m, 4H), 2.16 (t, J = 7.45, 2H,), 2.27 (t, J = 7.39, 2H), 2.52 (t, J = 6.64, 2H), 3.41 (t, J = 6.64, 2H), 3.67 (s, 3H). ¹³C NMR (75 MHz, CD3OD): 26.0, 26.9, 29.90, 29.92, 34.8, 34.9, 36.4, 36.9, 52.2, 173.9, 176.4, 177.7.

C₁₂H₂₁NO₅:MS (CI, NH3): m/z(%) 260(100, MH+), 277(41).

8-[2-(tert-Butoxycarbonylamino)ethylamino]-8-oxooctanoic acid (28)

Compound 26 (172 mg, 0.42 mmol) was dissolved in 5 mL MeOH. Pd-C (17 mg) was added and the mixture was stirred at 15 bar hydrogen pressure in an autoclave overnight. Pd/C was filtered off using celite and MeOH was evaporated. The product was obtained as a white solid (120 mg, 90%), m.p. 95 °C. ¹H NMR (300 MHz, CDCl3): 1.15 – 1.25 (m, 4H), 1.31 (s, 9H), 1.40 – 1.55 (m, 4H), 2.00 – 2.10 (m, 2H), 2.10 – 2.20 (m, 2H), 3.00 – 3.10 (m, 2H), 3.10 – 3.17 (m, 2H), 4.05 (bs, 3H). ¹³C NMR (75 MHz, CDCl3): 24.5, 25.3, 28.1, 28.5, 28.6, 33.9, 36.1, 39.6, 39.7, 79.6, 157.1, 174.9, 176.5. C15H28N2O5: MS (ES): m/z(%) 217(100), 261(45), 317(85, MH+), 339(15), 633(10), 650(25).

8-[3-(Benzyloxy)-3-oxopropylamino]-8-oxooctanoic acid (30)

Octanedioic acid (500 mg, 2.87 mmol), DIPEA (1.11 g, 8.61 mmol) and HOBT ⋅ H2O (427 mg, 3.16 mmol) were dissolved in ice-cold dichloromethane (20 mL) and EDC (490 mg, 3.16 mmol) was added under nitrogen atmosphere. After 15 min 3-aminopropanoic acid benzyl ester (617 mg, 2.87 mmol) was added and the ice bath was removed. The mixture was stirred overnight. Next day an aqueous NaHSO4 solution (5%w, 20 mL) was added and the crude product was extracted with dichloromethane (2 x 20 mL), the organic phase was dried over MgSO4, filtered and evaporated. The crude material was purified by column chromatography, (EA -> EA/MeOH 9:1). The obtained material is a 2:1 mixture of the desired product (420 mg, 44%) and the appropriate octanedioic acid diamide. The mixture was used in the next synthesis step without further purification.

tert-Butyl 2-(pyrene-1-carbonylamino)ethylcarbamate (32)

Pyrene-1-carboxylic acid (246 mg, 1.00 mmol), DIPEA (387 mg, 3.00 mmol) and HOBT ⋅ H₂O (149 mg, 1.10 mmol) were dissolved in ice-cold dichloromethane (20 mL). EDC (171 mg, 1.10 mmol) was added under nitrogen atmosphere. After 15 min Boc-protected ethylenediamine (160 mg, 1.0 mmol) was added and the ice bath was removed. The mixture was stirred overnight. Next day the reaction mixture was diluted with dichloromethane (40 mL), washed with aqueous NaHSO4 solution (5%w, 40 mL) and sat. aquoeus NaHCO3

solution (1 x 40 mL). The organic layer was dried over MgSO4 and evaporated. The crude compound was recrystallised from EA yielding the product as a yellow solid (213 mg, 55%), m.p. > 190 °C. ¹H NMR (300 MHz, DMSO-d6): 1.41 (s, 9H), 3.20 – 3.30 (m, 2H), 3.40 – 3.50 (m, 2H), 7.00 (t, J = 5.57, 1H), 8.05 – 8.30 (m, 5H), 8.30 – 8.40 (m, 3H), 8.50 – 8.54 (m, 1H), 8.71 (t, J = 5.37, 1H). ¹³C NMR (75 MHz, DMSO-d6): 28.2, 39.58, 39.61, 77.6, 123.5, 123.7, 124.2, 124.7, 125.3, 125.5, 125.7, 126.5, 127.1, 127.9, 128.2, 130.1, 130.6, 131.5, 131.8, 155.7, 168.9. C₂₄H₂₄N₂O₃: MS (EI): m/z(%) 201(58), 229(100), 245(57), 258(15), 389(22, MH+).

2-(Pyren-1-carbonylamino)ethanaminium chloride (33)

Compound 32 (549 mg, 1.41 mmol) was suspended in a mixture of 20 mL MeOH and 5 mL HCl (37%). It was stirred 30 min at 70 °C. The solvent was evaporated completely and a yellow solid was obtained (445 mg, 97%), m.p. > 190 °C. ¹H NMR (300 MHz, DMSO-d6):

3.05 – 3.20 (m, 2H), 3.65 – 3.75 (m, 2H), 8.05 – 8.15 (m, 1H), 8.15 – 8.30 (m, 3H), 8.30 – 8.35 (m, 4H), 8.35 – 8.50 (bs, 3H), 8.62 (m, 1H), 9.02 (t, J = 5.33, 1H). ¹³C NMR (75 MHz,

DMSO-d6): 37.3, 38.4, 123.5, 123.7, 124.2, 124.7, 125.6, 125.7, 125.8, 126.5, 127.1, 128.0, 128.3, 130.0, 130.6, 130.8, 131.7, 169.2. C₁₉H₁₆N₂O: MS (CI, NH3): m/z(%) 289(100, MH+).

3-Oxo-1-phenyl-2,5,8,11-tetraoxatridecan-13-oic acid (34)

3,6,9-Trioxaundecanedioic acid (5.55 g, 25 mmol), benzyl alcohol (2.70 mg, 25 mmol) and toluene sulfonic acid (43 mg, 0.25 mmol) were combined in a 250 mL flask and toluene (70 mL) was added. The flask was connected to a Dean-Stark apparatus. The mixture was heated to reflux and the reaction was completed after 1 h. Sat. aqueous NaHCO3 (50 mL) solution was added and the phases were separated. The aqueous layer was collected and extracted with EA (3 x 100 mL). The organic layer was dried over MgSO₄ and evaporated. The product is a viscous oil (1.87 g, 24%). ¹H NMR (300 MHz, CD₃OD): 3.60 – 3.75 (m, 8H), 4.10 (s, 2H), 4.20 (s, 2H), 5.17 (s, 2H), 7.30 – 7.40 (m, 5H). ¹³C NMR (75 MHz, CD₃OD): 66.7, 68.5, 68.6, 70.3, 70.5, 70.7, 70.9, 128.44, 128.47, 128.52, 128.63, 128.65, 135.3, 170.5, 173.2.

C15H20O7: MS (CI, NH₃): m/z(%) 242(6), 286(28), 313(2, MH+), 330(100).

Benzyl 1,6-dioxo-1-(pyren-1-yl)-8,11,14-trioxa-2,5-diazahexadecan-16-oate (35)

Compound 34 (462 mg, 1.48 mmol), DIPEA (476 mg, 3.69 mmol) and HOBT ⋅ H2O (200 mg, 1.48 mmol) were dissolved in DMF (10 mL) and EDC (229 mg, 1.48 mmol) was added (ice bath). After 15 min compound 33 (400 mg, 1.23 mmol) was added and the mixture was stirred overnight. Next day DMF was removed completely, the residue was dissolved in dichloromethane (20 mL) and washed with aqueous NaHSO4 solution (5%w, 20 mL), sat.

aqueous NaHCO3 solution (1 x 20 mL) and brine (1 x 20 mL). The crude product was purified with column chromatography (PE/EA 1:1 -> EA/MeOH 4:1 Rf = 0.3 [EA/MeOH 9:1]). The product is a yellow solid (420 mg, 59%), m.p. > 190 °C. ¹H NMR (300 MHz, CDCl3): 3.45 – 3.80 (m, 12H), 3.97 (s, 2H), 4.02 (s, 2H), 5.07 (s, 2H), 7.20 – 7.40 (m, 6H), 7.64 (t, J = 5.57, 1H), 7.95 – 8.10 (m, 6H), 8.14 – 8.19 (m, 2H), 8.55 – 8.60 (m, 1H). ¹³C NMR (75 MHz, CDCl3): 38.8, 40.9, 66.6, 68.4, 70.1, 70.27, 70.32, 70.7, 70.9, 124.3, 124.4, 124.6, 124.69, 124.7, 125.65, 125.72, 126.3, 127.1, 128.39, 128.47, 128.51, 128.55, 128.62, 130.7, 130.95, 131.12, 132.4, 135.2, 170.1, 170.4, 171.4. C34H34N2O7: MS (ES): m/z(%) 583(100, MH+), 605(60), 621(10). UV (MeOH): λ(ε) 233(65⋅10³), 242(89⋅10³), 265(34⋅10³), 275(51⋅10³), 326(31⋅10³), 340(41⋅10³). Fluorescence (MeOH): λ(type) 383 (monomer), 401 (monomer)

1,6-Dioxo-1-(pyren-1-yl)-8,11,14-trioxa-2,5-diazahexadecan-16-oic acid (36)

Compound 35 (203 mg, 0.35 mmol) was dissolved in 5 mL of MeOH. Pd/C (10 mg) was added and the mixture was stirred in an autoclave at 1 bar hydrogen pressure for 4 h. After that time Pd/C was filtered off with celite and the solvent was removed. The product is a yellow solid (172 mg, 100%), m.p. > 190 °C. ¹H NMR (300 MHz, CD3OD): 3.50 – 3.58 (m, 4H), 3.58 – 3.65 (m, 4H), 3.65 – 3.75 (m, 4H), 4.01 (s, 2H), 4.03 (s, 2H), 8.00 – 8.30 (m, 8H), 8.45 – 8.55 (m, 1H). ¹³C NMR (75 MHz, DMSO-d6): 38.1, 39.2, 67.5, 69.4, 69.6, 69.7, 69.9, 70.2, 123.5, 123.7, 124.3, 124.7, 125.2, 125.5, 125.7, 126.5, 127.1, 127.7, 128.0, 128.2, 130.1, 130.6, 131.5, 131.8, 169.0, 169.6, 171.6. C27H28N2O7: MS (ES): m/z(%) 493(100, MH+), 510(10), 515(50).

General procedure for the preparation of compounds 4b-4g, 4i, 4k-4q, 4t, 4u

The appropriate carboxylic acid (1 mmol), HOBT ⋅ H2O (1.2 mmol), EDC (1.2 mmol) and DIPEA (2 mmol) were combined in a flask under nitrogen atmosphere with 10 mL of cold DMF. After 15 min compound 3 (1 mmol) was added and the mixture was stirred overnight at room temperature. Next day the solvent was removed completely and the crude material was purified with column chromatography.

tert-Butyl (pentacosa-10,12-diynamido)methylthiomethylenecarbamate (4b)

Column chromatography: (PE/EA 9:1, Rf = 0.3). The product is a white solid (489 mg, 89%), m.p. 37 °C. ¹H NMR (300 MHz, CDCl3): 0.75 – 0.85 (t, J = 7.24, 3H), 1.10 – 1.70 (m, 41H), 2.17 (t, J = 6.92, 4H), 2.30 – 2.45 (m, 5H), 12.00 – 12.45 (m, 1H). ¹³C NMR (75 MHz, CDCl3): 14.13, 14.45, 19.18, 19.20, 22.69, 24.50, 27.99, 28.29, 28.36, 28.74, 28.86, 28.91, 29.06, 29.11, 29.35, 29.49, 29.62, 29.64, 29.65, 31.92, 37.38, 65.25, 65.34, 77.35, 77.54, 81.17, 160.97, 171.32, 171.49. C₃₂H₅₄N₂O₃S: MS (LC-MS-I): m/z(%) [tR = 15.3 min]:

547(100, MH+).

tert-Butyl (hexadecanamido)methylthiomethylenecarbamate (4c)

Column chromatography: (PE/EA 9:1 Rf = 0.5). The product is a white solid (342 mg, 80%), m.p. 62 °C. ¹H NMR (300 MHz, CDCl₃): 0.82 (t, J = 6.68, 3H), 1.18 – 1.28 (m, 24 H), 1.46 (s, 9H), 1.60 (m, 2H), 2.33 (s, 3H), 2.37 (m, 2H), 12.40 (bs, 1H). ¹³C NMR (75 MHz, CDCl₃): 14.1, 14.5, 28.0, 22.7, 29.1 – 29.7, 31.9, 80.2. C23H44N2O3S: MS (CI, NH₃): m/z(%) 329(15), 429(100, MH+).

tert-Butyl-[4-(pyren-1-yl)butanamido]methylthiomethylenecarbamate (4d)

Column chromatography (PE/EA 9:1 -> PE/EA 3:1, Rf = 0.4 [PE/EA 3:1]). The product was obtained as a white solid (299 mg, 65%), m.p. > 190 °C (decomp.). ¹H NMR (300 MHz, CDCl3): 1.53 (s, 9H), 2.18 – 2.32 (m, 2H), 2.32 – 2.43 (m, 3H), 2.45 – 2.70 (m, 2H), 3.41 (t, J

= 7.46, 2H), 7.80 – 7.90 (m, 1H), 7.95 – 8.05 (m, 3H), 8.05 – 8.20 (m, 4H), 8.25 – 8.35 (m, 1H), 12.15 – 12.55 (m, 1H). ¹³C NMR (75 MHz, CDCl3): 14.6, 26.1, 28.1, 32.5, 36.7, 81.3, 123.3, 124.86, 124.97, 125.01, 125.10, 125.88, 126.76, 127.32, 127.43, 127.47, 127.53, 128.74, 130.02, 130.03, 131.43, 135.26, 150.98, 161.04, 171.24. C27H₂₈N₂O₃S: MS (LC-MS- I): m/z(%) [tR = 15.5 min]: 461(100, MH+).

tert-Butyl-(pyren-1-carbonylamino)methylthiomethylenecarbamate (4e)

Column chromatography (PE/EA 9:1, R_f = 0.1). The product was obtained as a bright yellow solid (190 mg, 45%), m.p. > 190 °C (decomp.). ¹H NMR (300 MHz, CDCl₃): 1.54 – 1.63 (m, 9H), 2.53 – 2.67 (m, 3H), 7.95 – 8.40 (m, 7H), 8.90 – 9.00 (m, 1H), 9.40 – 9.50 (m, 1H), 12.60 – 13.40 (m, 1H). ¹³C NMR (75 MHz, CDCl₃): 15.1, 28.1, 83.6, 124.03, 124.36, 124.99, 125.55, 126.07, 126.21, 127.27, 127.38, 129.09, 129.54, 129.67, 130.42, 130.47, 131.05, 131.25, 134.25, 151.25, 171.15, 178.73. C24H22N2O3S: MS (LC-MS-I): m/z(%) [tR = 16.3 min]: 419(100, MH+), 837(10).

tert-Butyl 5,15,20-trioxo-20-(pyren-1-yl)-7,10,13-trioxa-2-thia-4,16,19-triazaicosan-3- ylidenecarbamate (4f)

Column chromatography (EA -> EA/MeOH 9:1, Rf = 0.3 [EA/MeOH 9:1]). The product was obtained as a light yellow solid (485 mg, 73%), m.p. > 190 °C (decomp.). ¹H NMR (300 MHz, CDCl3): 1.49 (s, 9H), 2.20 – 2.40 (m, 3H), 3.50 – 3.75 (m, 10H), 3.75 – 3.85 (m, 2H), 3.90 – 4.05 (m, 4H), 7.10 – 7.90 (m, 2H), 7.95 – 8.15 (m, 6H), 8.15 – 8.25 (m, 2H), 8.55 – 8.65 (m, 1H), 11.80 – 12.90 (m, 1H). ¹³C NMR (75 MHz, CDCl3): 14.4, 28.0, 39.0, 40.9, 70.4, 70.5, 70.6, 70.8, 70.9, 71.5, 77.2, 124.4, 124.6, 124.7, 125.7, 125.8, 126.3, 127.1, 128.6, 128.7, 130.7, 131.2, 132.5, 165.9, 169.1, 170.4. C₃₄H₄₀N₄O₈S: MS (ES): m/z(%) 565(50), 665(100, MH+), 687(15).

Benzyl 3-{8-[(Boc-amino)-methylthio-methyleneamino]-8-oxooctanamido}

propanoate (4g = 4o)^a

Column chromatography (PE/EA 1:1 -> PE/EA 3:7, Rf = 0.15[PE/ EA 1:1]). The product is a viscous oil (364 mg, 72%). ¹H NMR (300 MHz, CDCl₃): 1.25 – 1.40 (m, 4H), 1.51 (s, 9H),

1.55 – 1.70 (m, 4H), 2.05 – 2.15 (m, 2H), 2.35 – 2.50 (m, 2H), 2.38 (s, 3H), 2.55 – 2.60 (m, 2H), 3.45 – 3.55 (m, 2H), 5.13 (s, 2H), 6.02 (s, 1H), 7.30 – 7.40 (m, 5H), 12.45 (bs, 1H). ¹³C NMR (75 MHz, CDCl3): 14.5, 25.4, 28.0, 28.7, 34.1, 34.8, 36.6, 66.6, 77.3, 128.3, 128.4, 128.7, 135.6, 172.6, 172.9. C25H₃₇N₃O₆S: MS (ES): m/z(%) 408(25), 508(100, MH+).

a This compound was used for preparation of 1g and 1o.

(2R,3R,4S,5R)-6-[(Boc-amino)-methylthio-methyleneamino]-6-oxohexane-1,2,3,4,5- pentayl pentaacetate (4i)

Column chromatography (PE/EA 3:1, Rf = 0.1). Compound 4i (252 mg, 44%) was obtained as a viscous oil. ¹H NMR (300 MHz, CDCl3): 1.48 (s, 9H), 2.00 – 2.10 (m, 12H), 2.23 (s, 3H), 2.42 (s, 3H), 4.19 (dd, J1 = 5.84, J2 = 12.33, 1H), 4.34 (dd, J1 = 3.53, J2 = 12.33, 1H), 5.05 (dt, J1 = 3.53, J2 = 5.84, 1H), 5.40 (d, J = 3.08, 1H), 5.52 (dd, J = 5.84, 1H), 5.90 (dd, J1 = 3.08, J2 = 5.84, 1H), 11.88 (bs, 1H). ¹³C NMR (75 MHz, CDCl₃): 14.8, 20.5, 20.6, 20.7, 20.8, 21.1, 27.9, 61.5, 69.1, 69.3, 70.1, 74.5, 84.1, 150.5, 169.5, 169.7, 169.8, 170.2, 170.5, 174.5, 176.8. C23H34N2O13S: MS (ES): m/z(%) 579(100, MH+).

Methyl 3-{8-[boc-amino(methylthio)methyleneamino]-8-oxooctanamido}

propanoate (4k)

Column chromatography (PE/EA 1:1 -> EA, Rf = 0.4 [EA]). The product is a colorless oil (337 mg, 78%). ¹H NMR (300 MHz, CDCl3): 1.33 (m, 4H), 1.51 (s, 9H), 1.63 (m, 4H), 2.14 (m, 2H), 2.35 – 2.50 (m, 5H), 2.53 (m, 2H), 3.47 – 3.54 (dd, J1 = 6.07, J2 = 11.94, 2H), 3.70 (s, 3H), 6.04 (bs, 1H), 12.00 – 12.70 (bs, 1H). ¹³C NMR (75 MHz, CDCl3): 14.5, 24.3, 25.4, 28.0, 28.7, 28.8, 33.9, 34.7, 36.6, 37.3, 51.8, 77.2, 81.3, 171.3, 172.9, 173.3. C₁₉H₃₃N₃O₆S:

MS (CI, NH3): m/z(%) 332(14), 432(100, MH+).

tert-Butyl-{4-[4-(tert-butoxymethyl)-1H-1,2,3-triazol-1-yl]butanamido}

methylthio-methylenecarbamate (4l)

Column chromatography (PE/EA 3:1 -> PE/ EA 4:6, Rf = 0.4[PE/EA 4:6]). Compound 4l (254 mg, 62%) was obtained as a white solid, m.p. 93 °C. ¹H NMR (300 MHz, CDCl3): 1.26 (s, 9H), 1.49 (s, 9H), 2.15 – 2.30 (m, 2H), 2.37 (s, 3H), 2.40 – 2.60 (m, 2H), 4.40 (t, J = 6.33, 2H), 4.57 (s, 2H), 7.52 (s, 1H), 12.10 – 12.50 (m, 1H). ¹³C NMR (75 MHz, CDCl3): 14.6, 25.2, 27.6, 28.0, 35.3, 49.1, 56.5, 73.8, 81.5, 83.6, 122.1, 147.0, 171.0, 171.4. C18H31N5O4S:

MS (CI): m/z(%) 314(18), 414(100, MH+).

tert-Butyl {4-[4-(Boc-aminomethyl)-1H-1,2,3-triazol-1-yl]butanamido}

methylthio-methylenecarbamate (4m)

Column chromatography (PE/EA 3:1 -> PE/ EA 4:6, Rf = 0.3[PE/EA 4:6]). Compound 4m (341 mg, 75%) was obtained as a colorless, viscous oil. ¹H NMR (300 MHz, CDCl3): 1.34 (s, 9H), 1.41 (s, 9H), 2.10 – 2.25 (m, 2H), 2.29 (s, 3H), 2.30 – 2.50 (m, 2H), 4.25 – 4.40 (m, 4H), 5.20 – 5.40 (bs, 1H), 7.49 (s, 1H), 11.70 – 12.50 (m, 1H). ¹³C NMR (75 MHz, CDCl3): 14.5, 24.9, 27.9, 28.3, 33.9, 37.6, 49.4, 79.5, 122.0, 145.6, 155.9, 160.7, 169.9, 184.5.

C₁₉H₃₂N₆O₅S: MS (LC-MS-II): m/z(%) [tR = 7.3 min]: 457(100, MH+), 913(40).

((3aR,5R,5aS,8aS,8bR)-2,2,7,7-Tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-b:4',5'- d]pyran-5-yl)methyl 4-((boc-amino)-methylthio-methyleneamino)-4-oxobutanoate (4n) Column chromatography (PE/EA 9:1 -> PE/EA 3:1, R_f = 0.2 [PE/EA 9:1]). Compound 4n (396 mg, 74%) was obtained as a colorless, viscous oil. ¹H NMR (300 MHz, CDCl₃): 1.29 (d, J = 2.47, 6H), 1.40 (s, 3H), 1.42 – 1.50 (m, 12H), 2.34 (s, 3H), 2.60 – 2.85 (m, 4H), 3.90 – 4.00 (m, 1H), 4.19 (dd, J1 = 1.55, J2 = 7.85, 2H), 4.24 (d, J = 4.96, 1H), 4.28 (dd, J1 = 2.50, J2 = 4.99, 1H), 4.57 (dd, J1 = 2.46, J2 = 7.88, 1H), 5.48 (d, J = 4.98, 1H), 12.05- 12.49 (m, 1H). ¹³C NMR (75 MHz, CDCl3): 14.5, 24.5, 24.9, 25.9, 26.0, 27.9, 28.3, 29.3, 31.8, 35.8, 63.5, 63.8, 65.9, 70.4, 70.7, 71.0, 81.3, 83.4, 96.3, 108.7, 109.6, 150.9, 169.8, 170.9, 171.8, 172.8, 184.3. C23H₃₆N₂O₁₀S: MS (ES): m/z(%) 533(100, MH+).

tert-Butyl [17,17-dimethyl-13-(methylthio)-4,11,15-trioxo-16-oxa-3,12,14-triazaoctadec- 13-en-1-yl]carbamate (4p)

Column chromatography (PE/EA 1:1 -> PE/EA 3:7, Rf = 0.1 [PE/EA 1:1]). The product was obtained as a white solid (368 mg, 77%), m.p. 95 °C ¹H NMR (300 MHz, CDCl3): 1.25 – 1.35 (m, 4H), 1.37 (s, 9H), 1.40 – 1.50 (m, 9H), 1.50 – 1.65 (m, 4H), 2.05 – 2.15 (m, 2H), 2.32 (s, 3H), 2.32 – 2.50 (m, 2H), 3.15 – 3.35 (m, 4H), 4.80 – 4.95 (bs, 1H), 6.00 – 6.20 (bs, 1H), 12.00 – 12.50 (m, 1H). ¹³C NMR (75 MHz, CDCl3): 14.5, 24.3, 25.4, 28.0, 28.4, 28.6, 28.8, 36.5, 37.2, 40.3, 40.8, 79.7, 81.3, 157.0, 161.0, 171.3, 171.5, 173.7. C22H₄₀N₄O₆S: MS (ES): m/z(%) 389(18), 489(100, MH+).

tert-Butyl-16,16-dimethyl-5,14-dioxo-15-oxa-2-thia-4,11,13-triazaheptadecane-3,12- diylidenedicarbamate (4q)

Column chromatography (PE/EA 3:1, Rf = 0.35) Compound 4q (413 mg, 76%) was obtained as a white solid, m.p. 116-120 °C. ¹H NMR (300 MHz, CDCl₃): 1.30 – 1.45 (m, 2H), 1.48 (s,

9H), 1.49 (s, 9H), 1.52 (s, 9H), 1.55 – 1.75 (m, 4H), 2.30 – 2.55 (m, 5H), 3.35 – 3.45 (m, 2H), 8.30 (bs, 1H), 11.49 (bs, 1H), 12.16 – 12.48 (m, 1H). ¹³C NMR (75 MHz, CDCl3): 14.5, 24.2, 26.3, 28.0, 28.1, 28.3, 28.8, 37.1, 40.6, 79.2, 81.3, 83.1, 153.3, 156.1, 161.0, 163.6, 171.2, 171.3. C24H₄₃N₅O₇S: MS (LC-MS-I): m/z(%) [tR = 14.2 min]: 546(100, MH+), 1092(10).

Di-tert-butyl-17,17-dimethyl-5,8,15-trioxo-16-oxa-2-thia-4,9,12,14-tetraaza-octadecane- 3,13-diylidenedicarbamate (4t)

Column chromatography (PE/EA 4:6, Rf = 0.2). The product was obtained as a colorless, viscous oil (180 mg, 31%). ¹H NMR (300 MHz, CDCl3): 1.43 – 1.52 (m, 27H), 2.34 (s, 3H), 2.50 – 2.90 (m, 4H), 3.35 – 3.75 (m, 4H), 7.60 – 7.90 (m, 1H), 8.40 – 8.70 (m, 1H), 11.37 (bs, 1H), 11.75 – 12.50 (m, 1H). ¹³C NMR (75 MHz, CDCl₃): 14.5, 27.99, 28.04, 28.25, 38.6, 38.8, 40.4, 41.6, 79.6, 83.2, 83.6, 153.0, 153.1, 156.9, 157.4, 162.8, 177.5. C24H42N6O8S: MS (LC-MS-I): m/z(%) [t_R = 12.9 min]: 575(100, MH+), 1149(15).

tert-Butyl 14-boc-amino-12-boc-5,8-dioxo-2-thia-4,9,12-triazatetradecan-3- ylidenecarbamate (4u)

Column chromatography (PE/EA 1:1 -> EA, Rf = 0.15 [PE/EA 1:1]). The product was obtained as a colorless, viscous oil (386 mg, 67%). ¹H NMR (300 MHz, CDCl3): 1.40 (s, 9H), 1.45 (s, 9H), 1.48 (s, 9H), 2.35 (s, 3H), 2.40 – 2.90 (m, 4H), 3.10 – 3.50 (m, 8H), 4.85 – 5.10 (m, 1H), 6.50 – 7.00 (m, 1H), 11.70 – 12.60 (m, 1H). ¹³C NMR (75 MHz, CDCl3): 14.5, 28.0, 28.2, 28.3, 28.4, 39.0, 39.6, 47.2, 47.9, 79.4, 80.5, 156.1, 156.7, 170.5, 171.2.

C₂₅H₄₅N₅O₈S: MS (ES): m/z(%) 576(100, MH+).

tert-Butyl 11-boc-amino-5,8-dioxo-2-thia-4,9-diazaundecan-3-ylidenecarbamate (4s) DIPEA (0.37 g, 2.84 mmol) and TBTU (0.96 g, 3.0 mmol) were added to a solution of 22 (0.74 g, 2.84 mmol) in DMF (5 mL). The mixture was stirred under argon for 15 min prior to the addition of 3 (0.595 g, 3.13 mmol) and DIPEA (0.74 g, 2.84 mmol). After stirring over night glacial acetic acid (162 µL, 2.84 mmol) was added and the solvent was removed under reduced pressure (1 mbar) at a temperature of 45 °C. Purification with column chromatography (dichloromethane/EA 3:1 -> 1:2, Rf = 0.45[dichloromethane/EA 3:1]) yielded the product as a white solid (1.17 g, 95%). ¹H NMR (300 MHz, DMSO-d6): 1.37 (s, 9H), 1.42 (s, 9H), 2.27(2.25) (s, 3H), 2.30 – 2.38 (m, 2H), 2.53 – 2.59 (m, 2H), 2.09 – 2.99 (m, 2H), 2.99 – 3.10 (m, 2H), 6.75 – 6.90 (m, 1H), 7.86 – 7.99 (m, 1H), 11.24(10.98) (s, 1H).

C18H32N4O6S: (LC-MS-II): m/z(%) [t_R = 6.87 min]: 433(100, MH+).